Nitrate (NO₃¯) is an effective non‐protein nitrogen source for gut microbes and reduces enteric methane (CH₄) production in ruminants. Nitrate is reduced to ammonia by rumen bacteria with nitrite (NO₂¯) produced as an intermediate. The absorption of NO₂¯ can cause methaemoglobinaemia in ruminants. Metabolism of NO₃¯ and NO₂¯ in blood and animal tissues forms nitric oxide (NO) which has profound physiological effects in ruminants and has been shown to increase glucose uptake and insulin secretion in rodents and humans. We hypothesized that absorption of small quantities of NO₂¯ resulting from a low‐risk dose of dietary NO₃¯ will increase insulin sensitivity (SI) and glucose uptake in sheep. We evaluated the effect of feeding sheep with a diet supplemented with 18 g NO₃¯/kg DM or urea (Ur) isonitrogenously to NO₃¯, on insulin and glucose dynamics. A glucose tolerance test using an intravenous bolus of 1 ml/kg LW of 24% (w/v) glucose was conducted in twenty sheep, with 10 sheep receiving 1.8% supplementary NO₃¯ and 10 receiving supplementary urea isonitrogenously to NO₃¯. The MINMOD model used plasma glucose and insulin concentrations to estimate basal plasma insulin (Ib) and basal glucose concentration (Gb), insulin sensitivity (SI), glucose effectiveness (SG), acute insulin response (AIRg) and disposition index (DI). Nitrate supplementation had no effect on Ib (p > .05). The decrease in blood glucose occurred at the same rate in both dietary treatments (SG; p = .60), and there was no effect of NO³¯ on either Gb, SI, AIRg or DI. This experiment found that the insulin dynamics assessed using the MINMOD model were not affected by NO₃¯ administered to fasted sheep at a low dose of 1.8% NO₃¯ in the diet.

Nitrate (NO₃^–) supplementation is an effective methane (CH₄) mitigation strategy for ruminants but may produce nitrite (NO₂^–) toxicity. It has been reported that rumen protozoa have greater ability for NO₃^– and NO₂^– reduction than bacteria. It was hypothesised that the absence of ruminal protozoa in sheep may lead to higher NO₂^– accumulation in the rumen and a higher blood methaemoglobin (MetHb) concentration. An in vivo experiment was conducted with defaunated (DEF) and faunated (FAU) sheep supplemented with 1.8% NO₃^– in DM. The effects of rumen protozoa on concentrations of plasma and ruminal NO₃^– and NO₂^–, blood MetHb, ruminal volatile fatty acid (VFA) and ruminal ammonia (NH₃) were investigated. Subsequently, two in vitro experiments were conducted to determine the contribution of protozoa to NO₃^– and NO₂^– reduction rates in DEF and FAU whole rumen digesta (WRD) and its liquid (LIQ) and solid (SOL) fractions, incubated alone (CON), with the addition of NO₃^– or with the addition of NO₂^–. The results from the in vivo experiment showed no differences in total VFA concentrations, although ruminal NH₃ was greater (p < .01) in FAU sheep. Ruminal NO₃^–, NO₂^– and plasma NO₂^– concentrations tended to increase (p < .10) 1.5 hr after feeding in FAU relative to DEF sheep. In vitro results showed that NO₃^– reduction to NH₃ was stimulated (p < .01) by incoming NO₃^– in both DEF and FAU relative to CON digesta. However, adding NO₃^– increased (p < .05) the rate of NO₂^– accumulation in the SOL fraction of DEF relative to both fractions of FAU digesta. Results observed in vivo and in vitro suggest that NO₃^– and NO₂^– are more rapidly metabolised in the presence of rumen protozoa. Defaunated sheep may have an increased risk of NO₂^– poisoning due to NO₂^– accumulation in the rumen.